Despite its importance as the cause of a widely distributed, highly endemic form of cutaneous Leishmaniasis in the Old World, and as an increasingly recognized agent of visceral disease, studies on L. tropica have been neglected because of the absence of animal models. Metacyclic promastigotes of L. tropica were identified based on prior observations in L. donovani and L. major that the differentiation of promastigotes to an infective form is accompanied by structural modifications of the surface lipophosphoglycan (LPG). An anti-LPG monoclonal antibody (1H2A8) specific for L. tropica was used to negatively select for metacyclic forms in stationary cultures, and the exceptional virulence of the purified metacyclics was verified by their infectivity for mouse macrophages in vitro, and by their ability to produce cutaneous lesions in foot pads of BALB/c mice, and visceral disease in hamsters. The major finding from these preliminary animal studies is that both cutaneous and visceral clinical isolates of L. tropica produce similar cutaneous disease in BALB/c mice, but that the visceral isolates were much better able to disseminate to liver and spleen in the hamster model, suggesting that parasite intrinsic factors determine, at least in part, clinical outcome. We have initiated the first systematic study of the factors influencing the transmission of Leishmania by bite. The factors we are currently studying include the optimal time after the primary infective feed that the flies will refeed and egest an infectious inoculum, and whether prior sensitization of the host to sand fly saliva potentiates transmission. Preliminary studies clearly indicate that in mice previously bitten by uninfected sand flies, their subsequent exposure to saliva leads to a large influx of neutrophils, macrophages and dendritic cells into the dermis. The salivary molecules responsible for this inflammatory response, as well as the differences in the inflammatory response elicited by salivary components from different sand fly species, are currently being explored. Identifying the sand fly midgut receptor involved in promastigote adhesion using a ligand blotting technique and affinity purification have not been successful. We are attempting to clone the receptor by over-expression in mammalian cell lines. A cDNA library from unfed sand fly midgut was produced by PCR amplification of polyA+RNA using CapSwitch oligo and 3' CDS primers. Amplified material was size fractionated, pooled and cloned in a mammalian expression vector containing CMV promoter and SV40 sequence. CHO transfectants will be screened for LPG binding by flow cytometry.